Dehydrogenation of steroids by microorganisms of the genus mycococcus



This invention relates to a process for preparing A dehydrosteroids bythe use of microorganisms of the genus Mycococcus or enzyme produced bythem.

For a long time the present inventors have been searching for thosemicroorganisms which are able to form a double bond between thepositions 1 and 2 in ring A of steroids, thereby producing A-dehydrosteroids, and found that many microorganisms belonging to thegenus Mycococcus, have excellent ability to achieve the desired purpose.

The present invention has been accomplished based on the new finding andfurther studies and relates to a process for preparing A-dehydrosteroids which is characterized by bringing steroids belongingto the pregnane or androstane series, in which the positions 1 and 2 inthe steroid skeleton are saturated with hydrogen, into contact with theculture of microorganisms belonging to the genus Mycococcus, or with theoxidase produced by the microorganisms.

The starting materials used in the method of this invention are thosesaturated or unsaturated steroids belonging to the pregnane orandrostane series, in which the positions 1 and 2 in ring A aresaturated with hydrogen and positions other than 1 and 2 may besubstituted by an oxo, hydroxyl, halogeno, carboxyl, alkyl group or thelike, and the hydroxyl group may be protected in the form of ether orester and the oxo group in the form of ketal, hydrazone orsemicarbazone.

Typical ones of these steroids are as follows.

Pregnane-3,20-dione A -pregnadiene-3,20-dione A -pregnene-3,2O-dione-pregnen-11 (or 12,14,17 or 21 -l3,20-dione A -pregnen-14 (or 17 or21)-ol-3,11,20-trione A -pregnene-17,2l-diol-3,20-dione A -pregnene-17,21-diol-3,1 1,20-trione A -pregnene1l,2l-diol-3,20-trione A-pregnene-11,17,21-triol-3,20-dione A-pregnene-11,16,17,21-tetrol-3,20-dione 6 and/ or 9- (di)fiuoro-A-pregnene-1 1,17,2l-triol3,20-

dione 6-methyl-A -pregnene- 17,21-diol 3 ,1 1,20-trione A-pregnene-17,19,21-tri0l-3,20-dione A -pregnene-l1,20,2l-triol-3-oneAllopregnane-3,20-dione Androstane-3,17-dione A -androsten-l7-ol-3-one A(or A -androstene-3,l7-dione) A -androstene-3,11,17-trione or theirderivatives produced by changing their hydroxyl group into ester, ether,or halogenide and their oxo group into ketal, hydrazone, orsemicarbazone.

As mentioned before, many microorganisms belonging to the genusMycococcus can be used in the method of this invention, but it isdesirable to select those microorganisms which have excellent catalyticactivity for dehydrogenation.

Microorganisms especially suitable for the dehydrogenation of steroidsare as follows, for example.

Mycococcus sp. IFO No. 3574 States Patent Mycococcus sp. IFO No. 3588Mycoc ccus albus Krassilnikov Mycococcus capsulatus Krassilnikov Thenames of the genus of the above microorganisms are all based on BergeysManual of Determinative Bacteriology, 7th edition, published by TheWilliams & Wilkins Co., Baltimore, Md., U.S.A., in 1957. Thosemicroorganisms which lack in species name were separated by the presentinventors and deposited in Institute for Fermentation, Osaka, Osaka,Japan, under the number of IFO, respectively, and their microbialcharacteristics are as follows:

Mycococcus sp. lFO No. 3574- Cells round or ovoid, 1.0-1.2 microns indiameter, occurring singly, in pairs, in short chains, or in clumps.Sometimes rod-shaped cells also occur, particularly in liquid media.Non-motile. Grampositive.

Gelatin stab: No liquefaction.

Agar slant: Filiform, translucent, moist growth, be-

coming pale pink.

Broth: Faintly turbid.

Litmus milk: No visible change.

Potato: Moist pale pink growth.

Indole not produced.

Hydrogen sulfide not produced.

No acid and gas from glucose.

Starch not hydrolyzed.

Methyl red and Voges-Proskauer tests negative.

Citrates serve sole source of carbon.

Nitrites produced from nitrates.

Catalase-positive.

Aerobic.

Mycococcus sp. IFO No. 3588- Cells round or ovoid, 0.81.0 micron indiameter, occurring singly, in pairs, in short chains, or in clumps.Sometimes rod-shaped cells also occur, particularly in liquid media.Non-motile. Grampositive.

Gelatin stab: Crateriform liquefaction.

Agar slant: Filiform, translucent, moist growth.

Broth: F aintly turbid.

Litmus milk: Acid curd.

Potato: Slimy, pale orange-yellow streak.

Indole not produced.

Hydrogen sulfide produced.

Acid from glucose.

Starch not hydrolyzed.

Methyl red and Voges-Proskauer tests negative.

Citrates serve sole source of carbon.

Nitrites produced from nitrates.

Catalase-positive.

Aerobic.

In general, the incubation of microorganisms in this method is efiectedunder the conditions of oxidizing fermentation.

Nutrient media suitable for the growth of microorganisms contain carbonsource, nitrogen source assimilable by the microorganisms, and necessaryinorganic salts. As carbon source are used glucose, sucrose, dextrin,starch, and glycerin, for example and as nitrogen source are employednitrogen containing organic substances such as peptone, meat extract,casein, edamine, corn steep liquor, yeast, and yeast extract, organiccompounds such as amino acids, ammonium salts of organic acids, urea,and inorganic nitrogen compounds such as nitrates and ammonium salts.Necessary inorganic salts are potassium phosphate, sodium chloride,magnesium sulfate, etc., and the media may contain such metals ascopper, manganese, cobalt, and nickel. For a large scale run a liquidmedium is convenient.

The incubation of microorganisms may be effected statically but it ismore advantageous to conduct it under aerobic conditions such assubmerged culture under aeration with shaking or stirring.

Contact of the material steroids with the culture of microorganisms orwith their enzyme is effected by bringing the mycelium separated fromthe culture broth or the oxidase separated from the mycelium intocontact with the material steroids, or by adding the material steroidsto the medium at a proper stage of the incubation. In the latter case,the material is added at once or over a period as fine powder or as asolution or suspension in a suitable solvent such as methanol, ethanol,ethylene glycol, propylene glycol, dimethylformamide, dioxane, andwater, or as a solution or suspension containing a surface active agentor a dispersing agent. The pH of the substrate solution, incubationtemperature, incubation time, and other conditions are differentdepending on the kind and quantity of the starting steroids and the kindof microorganisms used, and therefore optimal conditions are selected ineach case. In general, however, the incubation is conducted at pH 6-9,25-30 C. for 3-50 hours, but these conditions are not necessarilyspecific.

According to the kind of microorganisms, their activities are different.If the incubation is carried out too long, using a strain of amicroorganism with strong activity, the A -dehydrosteroids onceaccumulated are decomposed resulting in a poor yield. Therefore, themost important factor for obtaining good yield of N-dehydrosteroids isincubation time, and in general less than 24 hours is preferable.

The A -dehydrosteroids thus accumulated in the culture broth can beseparated by various methods. For example, they are first adsorbed on aproper adsorbent such as alumina, magnesium silicate, or active carbonand then eluted with a suitable solvent such as methanol or ethanol, ordirectly extracted With a solvent immiscible With Water such aschloroform, methylene chloride, or ethylene chloride, or subjected tocounter current distribution. Or they are separated by chromatographyusing alumina, silica gel, cellulose, or pulp as carrier, or utilizingtheir difference in solubility in various solvents, or leading them totheir functional derivatives with Girard reagent T or P or with a loweraliphatic acid anhydride and a deacidating agent.

The products of the present invention, A -dehydrosteroids, are useful,for example, as medicines having activities of cortical hormones and/ orsexual hormones, or as intermediates for producing such medicines.

Example 1 A strain of Mycococcus sp. IFO No. 3574 is inoculated into 500cc. of nutrient medium constituted by a solution of 15 g. ofpolypeptone, 7.5 g. of meat extract, and 3 g. of KH PO in 1.5 l. of tapwater and adjusted to pH 7.0, in a 2 1. culture flask and incubated at28 C. for 24 hours with shaking. A solution of 500 mg. of A-pregnenedlp, 17a,2l-triol-3,20-dione in 20 cc. of dioxane is added andthe incubation is continued for additional 20 hours under the sameconditions. The culture broth is extracted four times With 500cc.-portions of ethyl acetate and the extract, after being washed With500 cc. of 1% sodium carbonate solution and 500 cc. of distilled watersuccessively, is evaporated under reduced pressure. The residue isdissolved in 100 cc. of 20% methanol and the solution, after beingWashed with 500 cc. of petroleum ether, is again extracted four timeswith 100 cc.-portions of ethyl acetate. The extract is dried overanhydrous sodium sulfate and concentrated under reduced pressure, andthe separated crystals are recrystallized from ethyl acetate to give 230mg. of A -pregnadiene-11,8,17a,2l-triol-3,20-dione.

The strain used in this example is deposited in Institute forFermentation, Osaka, and ATCC With numbers of IFO-3574 and ATCC-1355 6,respectively.

Example 2 A strain of Mycococcus sp. IFO No. 3574 is inoculated into 500cc. of the same nutrient medium as in Example 1 in each of three 2l.-culture flasks and incubated at 28 C. for 24 hours with shaking. Asolution of mg. of A pregnene-l 1 B,17a,2l-[IlOl-3,20-di0n6 in 4 cc. ofethanol is added to each medium and the incubation is continued foradditional 4 hours under the same conditions. The culture broth istreated in the same manner as in Example 1 to give 165 mg. of A-pregnadiene-1lB,17a,21-triol-3,20- dione.

Example 3 A 300 mg.-portion of A pregnene-ll/8,17a,21-triol-3, ZO-dioneis oxidized with a strain of Mycococcus sp. IFO No. 3588 and the sameconditions as in Example 1. The culture broths are combined andextracted four times with 300 cc.-portions of ethyl acetate and theextract is concentrated under reduced pressure to give a brown residue.The residue is dissolved in 100 cc. of methanol and the solution isdecolorized with 1.0 g. of active carhon and evaporated to give 186 mg.of n -pregnadiene- 11,8, 17a,2l-triol-3,20-dione.

The strain used in this example is deposited in Institute forFermentation, Osaka, and ATCC with members of IFO-3588 and ATCCl3557,respectively.

Example 4 A 500 mg.-portion of A -pregnene-l7a,2l-diol-3,2O-dione isoxidized with a strain of Mycococcus sp. IFO' No. 3574 under the sameconditions as in Example 2 and the reaction mixture is treated in thesame manner as in Example 3 to give mg. of A -pregnadiene-1704,21-diol-3,20-dione.

M-pregnene-17a,2l-diol-3,11,20-trione, A pregnene-1lp,21-diol-3,20-dione, A -pregnene-3,20-dione, or 90:- fluoro-Apregnene-l1,6,17a,2l-triol-3,20-dione can be oxidized in a similar Wayto the above to produce A -pregnadiene-l7a,2l-diol-3,l1,20-trione, A-pregnadiene-l1fl2 ldiol-3,20-dione, A -pregnadiene-3,20-dione or9u-fiuoron -pregnadiene-llfi,17u,21-triol-3,20-dione, respectively.

The above examples represent presently-preferred illustrativeembodiments of the invention, and in these examples, cc. stands forcubic centimeters, g. stands for grams, mg. for milligrams, and l. forliters.

Having thus disclosed the invention What is claimed is:

1. A process which comprises bringing a compound selected from the groupconsisting of steroids of the pregnane and androstane series, in whichat least the positions 1 and 2 are saturated, into contact with anenzyme system of an oxidase-producing microorganism of the genusMycococcus.

2. A process which comprises bringing a compound selected from the groupconsisting of steroids of the pregnane and androstane series, in whichat least the positions 1 and 2 are saturated, into contact with anenzyme system of a microorganism selected from the group consisting ofMycococcus sp. UFO-3574; ATCC13556) and Mycococcus sp. UFO-3588;ATCC-13557), thereby introducing a double bond between the positions 1and 2 of the steroid and producing the corresponding A -steroid.

3. The process claimed in claim 2, wherein the starting steroid compoundis selected from the group consisting of pregnane-3,20-dione, A-pregnadiene-3,20-dione, A pregnene-3,20-dione, A-pregnen-l1-ol-3,2O-dione, A pregnen-12-oll-3,20-dione, A-pregnen-l4-ol-3,20-dione, A pregnen-l7-ol-3,20-dione, A -pregnen-21-ol3,20 dione, A -pregnen-l4-ol-3,l1,20-trione, A -pregnen-17-ol-3,11,20-trione, A -pregnen-21-ol-3,11,20-trione, A -pregnene-l7,2l-diol-3,20-dione, A pregnene-17,2l-diol-3,l1,20-trione, A-pregnene-11,2l-diol-3,20-dione, M-pregnene-l 1,17,21- triol-3,20-dione,A -gpregnene-l1,16,l7,21-tetrol-3,20-dione, 6-(di)fluoro-A-pregnene-l1,l7,2l-triol-3,2O-dione, 9- (di)fluoro-A-pregnene-ll,l7,2l-triol-3,2O-dione, 6-1neth- 5 y1-A-pregnene-17,21-dil-3,11,2O-trione, M-pregnene- 17,19,21-trio1-3,20-di0ne, A -pregnene-11,20,21-trio1-3-one,a11opregnane-3,20-dione, androstane-3,17-dione, A-androsten-17-ol-3-0ne, A -andr0stene-2,17-dione, A -androstene-3,17-dione, and A -androstene-3,11,17-trione.

4. A process for converting A -pregnene-11p,17a,21- triol-3,20-dioneinto A -pregnadiene-11fi,17a,21-tri0l-3, 20-di0ne, which comprisesbringing the former into contact with an enzyme system of Mycococcus sp.(IPO- 3574; ATCC-13556), whereby a double bond is introduced between thepositions 1 and 2 to yield the said A -compound.

5. A process for converting A -pregnene-11B,17u,21- triol-3,20-dioneinto h -pregnadiene-l1fl,17a,21-trio1-3,

20-dione, which comprises bringing the former into contact 15 with anenzyme system of Mycococcus sp. (IFS-3558; ATCC-l3557), whereby a doublebond is introduced between the positions 1 and 2 to yield the said A-compound.

6. A process for converting A -pregnene-170:,21-di0l-3, -dione into N-pregnadiene-17a,2l-di0l3,20-dione, which comprises bringing the formerinto contact with an enzyme system of Mycococcus sp. UFO-3574); ATCC-13556), whereby a double bond is introduced between the positions 1 and2 to yield the said A -c0mpound.

References Cited in the file of this patent UNITED STATES PATENTS2,844,513 Wettstein et a1 July 22, 1958 2,905,592 Skull et al. Sept. 22,1959 2,908,696 Nussbaum et a1. Oct. 13, 1959 FOREIGN PATENTS 550,906Belgium Sept. 29, 1956

2. A PROCESS WHICH COMPRISES BRINGING A COMPOUND SELECTED FROM THE GROUPCONSISTING OF STERIODS OF THE PREGNANE ANDANDROSTANE SERIES,IN WHICH ATLEAST THE POSITIONS 1 AND 2 ARE SATURATED, INTO CNTACT WITH AN ENZYMESYMTEM OF A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OFMYCOCOCCUS SP. (IFO-3574; ATCC-13556) AND MYCOCOCCUS SP. (IFO-3588;ATCC-13557), THEREBY INTRODUCING A DOUBLE BOND BETWEEN THE POSITIONS 1AND 2 OF THE STERIOD AND PRODUCING THE CORRESPONDING $1-STEROID.